An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport

Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). Here, we show that orchestration of oskar RNP transport in the Drosophila germline relies on interplay between two double-stranded RNA-binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). We find that Staufen antagonizes Egl-mediated transport of oskar mRNA by dynein both in vitro and in vivo. Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. We additionally show that Egl associates with staufen (stau) mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. Our observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation of stau mRNA, and thus protein, in the oocyte enables motor switching on oskar RNPs by downregulating dynein activity.


Revision 0
Review #1 1. Evidence, reproducibility and clarity: Evidence, reproducibility and clarity (Required) **Summary:** It is well established that localization of oskar (osk) RNA in the Drosophila ovary proceeds in multiple steps. The first step depends upon dynein and results in delivery of osk into the oocyte. The second step involves kinesin-driven transport of osk to the oocyte posterior pole. The manuscript by Gáspár et al brings together several lines of evidence that support an antagonistic relationship with respect to motor binding between two osk-interacting proteins, Egalitarian (Egl) and Staufen (Stau). As staufen RNA and protein accumulate in the oocyte, Egl dissociates from osk, down-regulating dynein and enabling the second stage of osk transport to begin. **Major comments:** In general the experimental results support the conclusions drawn, and the paper includes a strong mix of in vitro and in vivo approaches. Nevertheless I have a few concerns.
(1)In Fig 1D it is apparent that stau KD increases the speed of both plus-end and minus-end runs to a highly significant degree, not just minus-end runs. The stimulating effect of loss of Stau on speed of plus-end runs is not mentioned in the text, and it perhaps muddies the argument that Stau is simply a negative regulator of dynein-dependent minus-end directed transport. This result needs to be explicitly discussed in the text.
(2)I recognize the importance of quantitative imaging to rigorously measure small differences in localization patterns. Nevertheless I find the data in Fig 3 extremely difficult to interpret. Presumably there is standard deviation everywhere there is green signal, but the magenta signal that corresponds to SD is not visible in most places that are green. I suggest adding to Fig 3 a single representative image for each genotype to illustrate each localization pattern, as well as a much clearer explanation of the quantitative imaging data. Perhaps the quantitative images could be moved to a supplemental figure.
**Minor comments:** (1)Color/density scales should be added to Figs 1A and S1A, otherwise the yellow/white signal at the posterior could be interpreted as something other than high abundance.
(2)In Fig 4A and 4C, I find it odd to have different halves of images photographed under different intensity settings and would prefer duplicate whole images.

Significance: Significance (Required)
The paper represents a substantial advance over existing knowledge and it extends our understanding about how RNAs can shuttle between different motor proteins to achieve a localized pattern. However, the Mohr et al 2021 PLoS Genetics paper covers some of the same ground. As that paper has now been published for several months, I believe a revised version of this paper should discuss that other work more prominently, making it apparent where the two studies concur and where this study extends the conclusions of the other one. If there are any contradictions between the two, those should be made explicit as well.

How much time do you estimate the authors will need to complete the suggested revisions: Estimated time to Complete Revisions (Required) (Decision Recommendation)
Less than 1 month Review #2 1. Evidence, reproducibility and clarity:

Evidence, reproducibility and clarity (Required)
In this manuscript, Gáspár et al. investigated the molecular mechanisms underlying the switching of motors for osk mRNA transport in the Drosophila ovary: from dynein in the nurse cells to kinesin-1 in the oocyte. They demonstrated that it requires two RNA-binding proteins, Egalitarian (Egl) and Staufen (Stau) to achieve the posterior localization of osk mRNA in the oocyte. Their data show that Egl is responsible for the stau mRNA transport into the oocyte, while Stau protein inhibits Egl-dependent dynein transport in the oocyte. Thus, they proposed a feed-forward mechanism in which Egl transports mRNA encoding its own antagonist Stau into the oocyte and thus achieves the switch of the osk mRNA transport from dynein to kinesin-1.
The antagonistic interaction between Egl and Staufen is well documented both in vitro and in vivo. All the results are carefully analyzed, but the data presentation is not reader-friendly. Overall, our main concern is about the role of Staufen in osk mRNA transport. **Here are specific points:** (1)According to the model, lack of Stau should result in failure of displacing Egl from the RNP complex and thus more dynein-driven transport in the oocyte. However, the increase of minusend run length in stau-RNAi is very small ( Figure 1E). It makes us wonder whether Stau is not a dominant inhibitor of Egl/dynein transport of osk RNPs. On the other hand, the speed increase of minus-end run in stau-RNAi is more dramatic than the run length ( Figure 1D-1E). Does it mean that in stau-RNAi dynein-driven osk transport has a shorter duration of run? Additionally, in Figure 1D, there is a statistically-significant increase of plus-end-directed transport velocity in stau-RNAi. While the author did mention that in the results "analysis of the speed and length of oskar RNP runs in ooplasmic extracts indicated that Khc activity was not compromised upon staufen knock-down", it does not explain the increased velocity towards the plus-end.
(2)What happened to osk mRNP transport in nurse cells with Staufen overexpression? The authors briefly mentioned that "GFP-Staufen overexpression has no major effect on the localization of oskar (Fig S1F-I)" on page 10. This is quite puzzling, as the authors propose that Staufen antagonized the Egl/dynein-driven transport. If the model holds true, we would expect to see that overexpression of Staufen causes less osk transport in nurse cells and thus less osk accumulated in the oocyte. Can the authors examine the osk mRNP transport in nurse cells in control and in GFP-Staufen overexpressing mutant and quantify the total amount of osk mRNA in the oocyte in control and after GFP-Staufen overexpression?
(3)Is osk mRNP transport in the nurse cells affected by stau-RNAi? The authors showed the Khc association with oskar mRNPs in the nurse cells in Figure 1C. We hope they could quantify the velocity and run length of the osk mRNP particles in nurse cells and compare control with stau-RNAi.
(4)The kymograms of in vitro motility assays (Figure 2A and Figure S2) clearly showed two different moving populations, fast and slow. Did the authors include both types of events in their quantifications? What are the N numbers for each quantification? What do the dots mean in Figure 2B-2G? Does each dot represent a single track in the kymograph? If so, we believe that the sample sizes are too small for in vitro motility assay.
(5)The in vitro motility assay showed that Staufen impairs dynein-driven transport of osk 5'-UTR ( Figure 2). Based on these data, it is unclear whether the effect of Staufen is osk mRNAdependent or Egl-dependent. We suggest performing the motility assay in the absence of osk 5'-UTR and Egl. Dynein, dynactin, and BicD should be sufficient to constitute the processive dynein complex in vitro. The addition of Staufen to the dynein complex will help to understand whether Staufen could directly affect dynein activity. We bring up this point because we noticed that the Staufen displacement of Egl in osk RNPs does not alter the amount of dynein complex associated ( Figure 6), implying that Staufen inactivates dynein activity on the RNP complex, independently of Egl-driven dynein recruitment.
(6)In Figure 4, it is hard to see any colocalization between GFP and osk mRNA. And the authors compared overexpressed Egl-GFP (driven by mat atub-Gal4 in mid-oogenesis) with Staufen-GFP under its endogenous promoter. An endogenous promoter-driven Egl-GFP would be much more appropriate for the comparison.
(7)In a recent publication (Mohr et al., 2021), a different model was proposed, in which Egl mediates transport, and Staufen facilitates the dissociation from the transport machinery for posterior anchoring. Although the authors referred to their paper in the discussion, they should acknowledge the differences and try to reconcile it (at least in the discussion).
(8)In the feed-forward model, Egl is required for the staufen mRNA transport from the nurse cells to the oocyte. Are Egl-GFP dots colocalized with staufen mRNAs in the nurse cells? Furthermore, to our understanding, in this model, the translation of the staufen mRNA would be critical for the switching motors between dynein and kinesin-1. In this sense, staufen mRNA translation is either suppressed in the nurse cells or only activated in the oocytes. I think the authors should at least address this point in the discussion. **Minor points:** 1)I hope the authors would show the osk mRNA localization in egl mutant in in individual stage 9 egg chambers. I can only find the osk mRNA in egl-RNAi early stage egg chambers ( Figure  7E), in which osk mRNA still shows an accumulation in the oocyte, although to a much lesser extent compared to control. In another publication (Sanghavi et al., 2016), it seems that the knockdown of Egl by RNAi causes some retention of osk mRNA in the nurse cells; but there are still noticeable amount of osk mRNA in the oocyte ( Figure 3A-B). We wonder whether the authors could quantify the amount of osk mRNA both in the nurse cells and in the oocyte of control and egl-RNAi. Also I wonder whether the authors could comment on fact that some osk mRNA transported into the oocyte. Could it be due to an egl-independent transport mechanism?
2)It is always nice to how the average distribution of osk mRNA (e.g., Figure 3, Figure S1, and Figure S3). But we recommend having a representative image of each genotype (a single egg) next to the average distribution. It will help the readers to better appreciate the differences among these genotypes.
3)The figure legends are overall hard to read and sometimes impossible to get information about the experiments (for example, Figure 4 legend). Can the authors improve their figure legends making them reader-friendly? 4)For moderate overexpression, the authors used P{matα4-GAL-VP16} (FBtp0009293). However, there are two different transgenic lines associated with FBtp0009293 (V2H and V37), which have slightly different expression levels. The authors should specify which line they used in the experiments. 5)On page 13 "PCR on egg-chambers co-expressing Egl-GFP and either staufen RNAi or a control RNAi (white) in the germline (Fig 3G)", it should be Figure 4G.

Evidence, reproducibility and clarity (Required)
Some additional experimental evidence is needed to solidify the conclusions and provide definitive support for this model, as discussed below.
Biochemical experiments using UV crosslinking and GFP immunoprecipitation followed by quantitative PCR were performed to show that Staufen antagonizes the association of Egl with oskar mRNA in vivo. -The authors need to show the quantitative analysis, which was not present in the figure, specifically the effects of Staufen RNAi compared to control.
Is the ability of Staufen to antagonize and displace Egl dependent on Staufen binding to Oscar RNA? Will a Staufen mutant that can't bind to RNA also displace Egl? Alternatively, the mechanism may be independent of RNA binding and perhaps due to protein-protein interactions.
A key question addressed is how does Staufen play a role in directing Oscar RNA localization to the posterior pole. The spatiotemporal control of Staufen at stage 9 seems to be a critical step. A number of experiments are performed to show that Staufen RNA enters the oocyte and accumulates to anterior pole through a process dependent on Egl (Fig. 7).
-Definitive evidence is needed to show the role of 3'UTR of Stau and Egl binding. As it stands now, no evidence is presented to prove that delivery of staufen RNA via Egl, rather than dumping of Staufen protein into oocytes is the necessary trigger for the switch. It is well known that Staufen protein is also transported through ring canals to deliver Staufen into oocytes. There is no need to invoke an additional mechanism of Egl mediated staufen mRNA delivery. A key experiment is to perturb the Egl interaction with staufen 3'UTR and show this is a necessary component to impact oscar. Related to this comment, they should first perform biochemistry IP and PCR to demonstrate association of Egl with staufen RNA, and then somehow perturb this interaction to assess effects on oscar RNA localization. For example, is the 3'UTR of staufen RNA necessary for this mechanism? What if staufen RNA was ectopically localized in some inappropriate manner, for example localized to posterior pole? Would this prevent the switch of oscar RNA to move to posterior pole? The key question is: is it necessary that translation of Stau be coupled to Egl in order to drive the switch. **Minor comments** "Substantially more oskar mRNA was co-immunoprecipitated with Egl-GFP f rom extracts of egg-chambers expressing staufen RNAi compared t o t he control (Fig 3G). -This data is not shown in 3G, but rather only in Fig. S4H which needs quantitative analysis shown.
"Addition of recombinant Staufen to the Egl, BicD, dynein and dynactin assembly mix significantly reduced the number of oskar mRNA transport events ( Fig. 2A and B)." -In Fig. 2A, the Y axis shows velocity not number of transport events Staufen may also be required for the efficient release of the mRNA from the anterior cortex. This may reflect a role of Staufen in the coupling of the mRNA to the kinesin-dependent posterior transport pathway. This could be discussed as another aspect of the inhibition of dynein and handoff to kinesin.

Significance (Required)
This elegant manuscript by Gaspar et al provides new insight into the spatiotemporal regulation of Staufen mediated localization of oscar mRNA to the posterior pole in Drosophila oocytes.
Here the authors demonstrate the competitive displacement of the RNA binding protein Egalitarian, which antagonizes dynein dependent localization at the anterior pole. This work done in this well characterized model of mRNA localization in Drosophila oocytes has broader implications for how the bidirectional transport of mRNAs is regulated in other polarized and highly differentiated cells, where very little is know about how mRNA transport direction might be regulated by opposing activities of kinesin and dynein motors. The strengths of this study are the integration of microscopy, biochemisty and genetic mutants to provide very nice experimental support for the two major aspects to the proposed model: 1) the competition between Staufen and Egl on oscar RNA which affects localization, 2) evidence for Egl mediated localization of staufen RNA into the oocyte as a key trigger for competitive displacement to bias localization of oscar RNA via kinesin. However, some additional experimental evidence is needed to solidify the conclusions and provide definitive support for this model, as discussed in other section.

How much time do you estimate the authors will need to complete the suggested revisions: Estimated time to Complete Revisions (Required) (Decision Recommendation)
Less than 1 month

General Statements [optional]
We are very grateful to the reviewers for their constructive comments. In response to their critiques, we have made extensive modifications to the manuscript, including documenting new experiments and analyses, and improving data presentation. Here we provide a point-by-point response to the reviewers' comments. We apologize for the delay in resubmitting, which was due to the first (and co-corresponding) author moving to a different position.

Point-by-point description of the revisions
Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary:
It is well established that localization of oskar (osk) RNA in the Drosophila ovary proceeds in multiple steps. The first step depends upon dynein and results in delivery of osk into the oocyte. The second step involves kinesin-driven transport of osk to the oocyte posterior pole. The manuscript by Gáspár et al brings together several lines of evidence that support an tantagonistic relationship with respect to motor binding between two osk-interacting proteins, Egalitarian (Egl) and Staufen (Stau). As staufen RNA and protein accumulate in the oocyte, Egl dissociates from osk, down-regulating dynein and enabling the second stage of osk transport to begin.

Major comments:
In general the experimental results support the conclusions drawn, and the paper includes a strong mix of in vitro and in vivo approaches. Nevertheless I have a few concerns.
(1)In Fig 1D it is apparent that stau KD increases the speed of both plus-end and minus-end runs to a highly significant degree, not just minus-end runs. The stimulating effect of loss of Stau on speed of plus-end runs is not mentioned in the text, and it perhaps muddies the argument that Stau is simply a negative regulator of dynein-dependent minus-end directed transport. This result needs to be explicitly discussed in the text.
We thank the reviewer for this important comment. Indeed, our previous analysis of the overall population of oskar RNPs showed that plus-end-directed runs had increased velocity in the absence of Staufen (although the magnitude of the effect was considerably smaller than Full Revision observed for minus-end-directed runs). The reviewer's comment prompted us to analyze the effects on motility in more detail. In particular, we have now stratified the data based on the RNA content of the RNPs to control for effects of Staufen depletion on RNA copy number of the motile oskar RNPs. These analyses, which are documented in Fig 1B-F of the revised manuscript and discussed between lines 103 and 141 (merged PDF file), indicate that the previous velocity and run length data was somewhat confounded by the Staufen-depleted condition having a lower fraction of moving complexes with a large RNA content, which generally move more slowly. Accounting for this effect shows that impairing Staufen has no significant effect on plus-end-directed run lengths, whereas minus-end-directed run lengths are substantially increased. The velocity of runs is also specifically increased in the minus-end direction in the Staufen-depleted background for RNPs that have a relative RNA content of 1 or 2 units, which represent the majority of the RNP population in that genotype. Whilst RNPs with larger RNA content (2 relative units) do have significantly higher plus-end-directed velocity compared to the same category in the control, the effect is of much smaller magnitude than observed for minus-end-directed movements by this population. To help clarify these results, magnitudes of the effects are now shown in the new Fig. 1

E and F.
These data strengthen the case that Staufen predominantly affects minus-end-directed motion. Given many documented examples of the interdependence of dynein and kinesin on bidirectional cargoes (Hancock et al. 2014), it is conceivable that the modest effects on plusend-directed velocity for a subset of RNPs arise indirectly from the influence of Staufen on dynein activity. However, we agree with the reviewer that we should not rule out the alternative possibility that Staufen has additional roles in regulating oskar transport, including potentially modulating kinesin-1 directly. We have therefore added a section to the Discussion that covers this issue (lines 503-521).
(2) I recognize the importance of quantitative imaging to rigorously measure small differences in localization patterns. Nevertheless I find the data in Fig 3 extremely Presumably there is standard deviation everywhere there is green signal, but the magenta signal that corresponds to SD is not visible in most places that are green. I suggest adding to Fig 3 a single representative image for each genotype to illustrate each localization pattern, as well as a much clearer explanation of the quantitative imaging data. Perhaps the quantitative images could be moved to a supplemental figure.
Reviewer 2 also suggested that we include representative images in addition to the quantitative readout. We have now replaced the old Figure 3

Full Revision
(1)Color/density scales should be added to Figs 1A and S1A, otherwise the yellow/white signal at the posterior could be interpreted as something other than high abundance.
We thank the reviewer for spotting this. We have now added a color scale to the relevant figures.
(2)In Fig 4A and 4C, I find it odd to have different halves of images photographed under different intensity settings and would prefer duplicate whole images.
We used this layout to illustrate in the most compact way possible the (co)localization of the two RBPs and oskar RNA in the nurse cell and oocyte compartments, where signal intensities can differ dramatically. Following the reviewer's comment, we now show whole images with different intensity settings (Figure 4 A, A', C, C'). We thank the reviewer for spotting this error, which has now been corrected.

Reviewer #1 (Significance (Required)):
The paper represents a substantial advance over existing knowledge and it extends our understanding about how RNAs can shuttle between different motor proteins to achieve a localized pattern. However, the Mohr et al 2021 PLoS Genetics paper covers some of the same ground. As that paper has now been published for several months, I believe a revised version of this paper should discuss that other work more prominently, making it apparent where the two studies concur and where this study extends the conclusions of the other one. If there are any contradictions between the two, those should be made explicit as well.
We had discussed the Mohr et al. study in our manuscript, which came out when our work was in preparation. Following the reviewer's comment, we now address explicitly how our study differs from theirs and how our work extends their findings. The relevant paragraphs in the Discussion begin on lines 442 and 503. Briefly, a key point of difference is that Mohr et al. focused on the Transport and Anchoring Sequence (TAS) (including its ability to associate with Egl) and other Staufen recognition sites (SRSs) in oskar mRNA. Their study also includes an experiment examining the effect of Egl overexpression on oskar localization (as described in our original submission). In contrast, our study directly examines the interplay between the RBPs Staufen and Egl on oskar RNPs. We are the first to show that Staufen directly antagonizes dynein-based transport and that this is associated, at least in part, with an ability to impair Egl association with RNPs. Moreover, we provide insights into the in vivo role of Egl/BicD in recruitment vs activation of dynein on RNPs and how the activity of Staufen is coordinated in space and time via Egl-mediated delivery of stau mRNA, which constitutes a novel type of feedforward mechanism. We do not believe there are any contradictions between the two studies.

Full Revision
Reviewer #2 (Evidence, reproducibility and clarity (Required)): In this manuscript, Gáspár et al. investigated the molecular mechanisms underlying the switching of motors for osk mRNA transport in the Drosophila ovary: from dynein in the nurse cells to kinesin-1 in the oocyte. They demonstrated that it requires two RNA-binding proteins, Egalitarian (Egl) and Staufen (Stau) to achieve the posterior localization of osk mRNA in the oocyte. Their data show that Egl is responsible for the stau mRNA transport into the oocyte, while Stau protein inhibits Egl-dependent dynein transport in the oocyte. Thus, they proposed a feed-forward mechanism in which Egl transports mRNA encoding its own antagonist Stau into the oocyte and thus achieves the switch of the osk mRNA transport from dynein to kinesin-1.
The antagonistic interaction between Egl and Staufen is well documented both in vitro and in vivo. All the results are carefully analyzed, but the data presentation is not reader-friendly. Overall, our main concern is about the role of Staufen in osk mRNA transport. **Here are specific points:** (1)According to the model, lack of Stau should result in failure of displacing Egl from the RNP complex and thus more dynein-driven transport in the oocyte. However, the increase of minusend run length in stau-RNAi is very small ( Figure 1E). It makes us wonder whether Stau is not a dominant inhibitor of Egl/dynein transport of osk RNPs. On the other hand, the speed increase of minus-end run in stau-RNAi is more dramatic than the run length ( Figure 1D-1E). Does it mean that in stau-RNAi dynein-driven osk transport has a shorter duration of run? Additionally, in Figure 1D, there is a statistically-significant increase of plus-end-directed transport velocity in stau-RNAi. While the author did mention that in the results "analysis of the speed and length of oskar RNP runs in ooplasmic extracts indicated that Khc activity was not compromised upon staufen knock-down", it does not explain the increased velocity towards the plus-end.
We thank the reviewer for these insightful comments.
We and others (Zimyanin et al. 2008; Gaspar et al., 2014) have shown that there is only a small posterior-directed bias in oskar RNP transport in the wild-type ooplasm at mid-oogenesis. Thus, small increases in minus-end-directed transport parameters are expected to be sufficient for anterior mislocalization of a subset of RNPs, as is seen in stau mutants (note that we would not expect a dramatic increase in minus-end-directed motile properties in the stau RNAi condition, as a significant fraction of oskar RNA is targeted posteriorly). To allow the readers to better judge the magnitude of the effects, we now include the percentage change in mean velocity and run length values on the graphs (new Figure 1E and F).
Regarding the reviewer's question about the run duration, indeed it is shorter for the minus-end directed runs in the absence of Staufen. In the motor field, it is typical to present velocity and run length only because duration is dependent on these two parameters.
Reviewer 1 also made a similar comment about plus-end directed velocity of RNPs. As we wrote in response to their comment, we have now stratified the data based on the RNA content of the RNPs to control for effects of Staufen depletion on RNA copy number of the motile oskar RNPs. These analyses, which are documented in Fig 1 B-F of the revised manuscript and discussed between lines 103 and 141, indicate that the previous velocity and run length data were somewhat confounded by the Staufen-depleted condition having a lower fraction of moving complexes with a large RNA content, which generally move more slowly. Accounting for this effect shows that impairing Staufen has no significant effect on plus-end-directed run lengths, whereas minus-end-directed run lengths are substantially increased. The velocity of runs is also increased only in the minus-end direction in the Staufen-depleted background for RNPs that have a RNA content of 1 or 2 relative units, which represent the majority of the RNP population in that genotype. Whilst RNPs with larger RNA content (2 relative units) do have significantly higher plus-end-directed velocity compared to the same category in the control, the effect is of much smaller magnitude than observed for minus-end-directed movement for this population.
These data strengthen the case that Staufen predominantly affects minus-end-directed motion. Given many documented examples of the interdependence of dynein and kinesin on cargoes (Hancock et al., 2014), it is conceivable that the modest effects on plus-end-directed velocity arise indirectly due to the influence of Staufen on dynein activity. However, we agree with the reviewer that we should not rule out the alternative possibility that Staufen has additional roles in regulating oskar transport, including potentially modulating kinesin-1 activity directly. We have therefore added a section to the Discussion that covers this issue (lines 503-521).
(2) What happened to osk mRNP transport in nurse cells with Staufen overexpression? The authors briefly mentioned that "GFP-Staufen overexpression has no major effect on the localization of oskar (Fig S1F-I)" on page 10. This is quite puzzling, as the authors propose that Staufen antagonized the Egl/dynein-driven transport. If the model holds true, we would expect to see that overexpression of Staufen causes less osk transport in nurse cells and thus less osk accumulated in the oocyte. Can the authors examine the osk mRNP transport in nurse cells in control and in GFP-Staufen overexpressing mutant and quantify the total amount of osk mRNA in the oocyte in control and after GFP-Staufen overexpression?
We showed in the initial submission that strong overexpression of GFP-Staufen in early oogenesis (e.g. with osk-Gal4) disrupts oskar localization, including causing ectopic accumulation in the nurse cells (Fig S7F and G, now marked with arrowheads). Fig S1F-I, to which the reviewer refers, documents an experiment in which the expression of GFP-Staufen was directly driven by the maternal tubulin promoter (i.e. not through the UAS-Gal4 system; now indicated in Fig. S1F). We had assumed that the difference in behavior of the different GFP- (3)Is osk mRNP transport in the nurse cells affected by stau-RNAi? The authors showed the Khc association with oskar mRNPs in the nurse cells in Figure 1C. We hope they could quantify the velocity and run length of the osk mRNP particles in nurse cells and compare control with stau-RNAi.

Full Revision
We have never succeeded in making squashes of nurse cells that maintain oskMS2 RNA transport. Therefore, we are unable to evaluate directional transport of oskar in these cells. However, Staufen does not accumulate to appreciable levels in the nurse cells, as shown by Little et al., 2015 and also Figure 4A and A' (left panels). Moreover, we did not detect significant colocalization between Staufen and oskar in the nurse cells (Fig. 4B). Therefore, depletion of Staufen with RNAi is not expected to influence motility of oskar in this part of the egg chamber.
(4)The kymograms of in vitro motility assays (Figure 2A and Figure S2) clearly showed two different moving populations, fast and slow. Did the authors include both types of events in their quantifications? What are the N numbers for each quantification? What do the dots mean in Figure 2B-2G? Does each dot represent a single track in the kymograph? If so, we believe that the sample sizes are too small for in vitro motility assay.
For completeness, we did not exclude particles from our analysis based on their speed of movement. We have now made this point clear in an updated section of the Methods (lines 807-811), which provides additional information on particle inclusion criteria.
We did document in the legends what the dots represent (values for single microtubules). We have now also included information on the number of complexes analyzed, which is 586-1341 single RNA particles or 1247-2207 single dynein particles per condition. These sample sizes are considerably larger than those used in most in vitro motility studies.
(5)The in vitro motility assay showed that Staufen impairs dynein-driven transport of osk 5'-UTR ( Figure 2). Based on these data, it is unclear whether the effect of Staufen is osk mRNAdependent or Egl-dependent. We suggest performing the motility assay in the absence of osk 5'-UTR and Egl. Dynein, dynactin, and BicD should be sufficient to constitute the processive dynein complex in vitro. The addition of Staufen to the dynein complex will help to understand whether Staufen could directly affect dynein activity. We bring up this point because we noticed that the Staufen displacement of Egl in osk RNPs does not alter the amount of dynein complex Full Revision associated (Figure 6), implying that Staufen inactivates dynein activity on the RNP complex, independently of Egl-driven dynein recruitment.
We cannot look at transport of dynein in the presence of only dynactin and full-length BicD as BicD is not activated (and thus unable to effectively bind dynein and dynactin) without . We find that Staufen partially inhibits DDB motility but not to the extent seen with the full-length BicD in the presence of Egl and RNA (new main figure panels 2H and I, and Figure S3). As stated on lines 186 -188, these data suggest that Staufen inhibits both the activation of dynein-dynactin motility by BicD proteins, as well as stimulation of this event by Egl and RNA. This finding is also incorporated in a new section of the Discussion that covers possible roles of Staufen in addition to competing for Egl's binding to RNA (between lines 503-521). We are very grateful to the reviewer for suggesting this approach, as it has provided significant new insight into Staufen's function.
(6)In Figure 4, it is hard to see any colocalization between GFP and osk mRNA. And the authors compared overexpressed Egl-GFP (driven by mat atub-Gal4 in mid-oogenesis) with Staufen-GFP under its endogenous promoter. An endogenous promoter-driven Egl-GFP would be much more appropriate for the comparison.
Colocalization between GFP and oskar signals is seen as white in Fig. 4A and C. We have now added arrows to highlight a few examples of colocalization. The degree of colocalization was quantified in an unbiased fashion (shown in panels Fig 4B and D).
Regarding the expression of Egl-GFP: it was driven directly by the aTub84B promoter and not by matTub-Gal4. Western blot analysis performed in response to the reviewer's comment shows that Egl-GFP is expressed at similar levels to endogenous Egl in this line (new Fig. S5I).
(7)In a recent publication (Mohr et al., 2021), a different model was proposed, in which Egl mediates transport, and Staufen facilitates the dissociation from the transport machinery for posterior anchoring. Although the authors referred to their paper in the discussion, they should acknowledge the differences and try to reconcile it (at least in the discussion).
We now further discuss our work in the light of the findings by Mohr et al. (a request also made by Reviewer 1) (in paragraphs starting on lines 442 and 503). In our opinion, the data of Mohr et al. in fixed material cannot discriminate between effects of Staufen (or the TAS) on transport vs anchorage. In contrast, our dynamic imaging in vitro and ex vivo shows unambiguously that Staufen can modulate transport processes. As accumulation of RNA at the cortex is dependent on directional transport, we do not think it necessary to invoke a separate anchorage role of Staufen. We have now raised the possibility that transport and cortical localization are two Full Revision facets of the same underlying process in the hope that this will stimulate further investigation (lines 462-466).
(8)In the feed-forward model, Egl is required for the staufen mRNA transport from the nurse cells to the oocyte. Are Egl-GFP dots colocalized with staufen mRNAs in the nurse cells?
We showed in Fig 7I of the original submission that Egl-GFP puncta are colocalized with stau mRNAs in nurse cells. Indeed, this is a key piece of evidence for our model. These data are now in Figure 7F.
Furthermore, to our understanding, in this model, the translation of the staufen mRNA would be critical for the switching motors between dynein and kinesin-1. In this sense, staufen mRNA translation is either suppressed in the nurse cells or only activated in the oocytes. I think the authors should at least address this point in the discussion. This is another excellent suggestion. We have now included in the Discussion (from line 533) the point that Staufen translation may be suppressed during transit to the oocyte or that the protein may be translated en route but only build up to meaningful levels where the RNA is concentrated in the oocyte. **Minor points:** 1)I hope the authors would show the osk mRNA localization in egl mutant in in individual stage 9 egg chambers. I can only find the osk mRNA in egl-RNAi early stage egg chambers ( Figure 7E), in which osk mRNA still shows an accumulation in the oocyte, although to a much lesser extent compared to control. In another publication (Sanghavi et al., 2016), it seems that the knockdown of Egl by RNAi causes some retention of osk mRNA in the nurse cells; but there are still noticeable amount of osk mRNA in the oocyte ( Figure 3A-B). We wonder whether the authors could quantify the amount of osk mRNA both in the nurse cells and in the oocyte of control and egl-RNAi. Also I wonder whether the authors could comment on fact that some osk mRNA transported into the oocyte. Could it be due to an egl-independent transport mechanism? egl null mutants do not reach stage 9 due to a defect in retention of oocyte fate, hence the use of egl RNAi in our study and the one by Sanghavi et al. Whilst we can't rule out a (minor) Eglindependent mechanism for localizing oskar RNA in the oocyte, to date no other pathway has been implicated in the delivery of this or any other mRNA from the nurse cells. We favor a scenario in which residual oskar accumulation in the oocyte in egl RNAi egg chambers is due to incomplete depletion of Egl protein in the knockdown condition. We have noted this in the relevant figure legend and also clarify that the RNAi is a tool for knockdown in line 396 of the Results section.

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The below plot shows a quantification of oskar mRNA localization in egl and control RNAi egg chambers, which the reviewer was wondering about.
In the egl RNAi egg-chambers, there is a significant increase in the mean signal intensity of 2)It is always nice to how the average distribution of osk mRNA (e.g., Figure 3, Figure S1, and Figure S3). But we recommend having a representative image of each genotype (a single egg) next to the average distribution. It will help the readers to better appreciate the differences among these genotypes.
This suggestion was also made by Reviewer 1. We have added representative images to Figure  3 and moved the images depicting average distributions to the supplement (Fig S4). We have also improved the legend and labeling for Fig S4.

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3)The figure legends are overall hard to read and sometimes impossible to get information about the experiments (for example, Figure 4 legend). Can the authors improve their figure legends making them reader-friendly?
We have edited the legends to make them clearer, including an extensive reworking of those for Figure 4. We thank the reviewer for encouraging us to do this. 4)For moderate overexpression, the authors used P{matα4-GAL-VP16} (FBtp0009293). However, there are two different transgenic lines associated with FBtp0009293 (V2H and V37), which have slightly different expression levels. The authors should specify which line they used in the experiments.
The matTub-Gal4 transgene we used in our study is inserted in the 2nd chromosome. We now mention this in the Methods section (line 574). We received this line from another lab many years ago, with no additional information provided. 5) On page 13 "PCR on egg-chambers co-expressing Egl-GFP and either staufen RNAi or a control RNAi (white) in the germline (Fig 3G)", it should be Figure 4G.
We apologize for this mistake, which has now been fixed.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Some additional experimental evidence is needed to solidify the conclusions and provide definitive support for this model, as discussed below.
Biochemical experiments using UV crosslinking and GFP immunoprecipitation followed by quantitative PCR were performed to show that Staufen antagonizes the association of Egl with oskar mRNA in vivo. -The authors need to show the quantitative analysis, which was not present in the figure, specifically the effects of Staufen RNAi compared to control.
These quantitative data, which are key for our model, were shown in the original submission ( Fig 4G in the original and revised manuscript). We mistakenly called out the panel as 3G in the original submission. We apologize for this error, which has now been dealt with.
Is the ability of Staufen to antagonize and displace Egl dependent on Staufen binding to Oscar RNA? Will a Staufen mutant that can't bind to RNA also displace Egl? Alternatively, the mechanism may be independent of RNA binding and perhaps due to protein-protein interactions.
While the details of how Staufen displaces Egl are certainly an interesting topic for future research, we consider that addressing this goes well beyond the scope of this study, which already covers a lot of ground. Staufen contains four double stranded RNA-binding domains, and deleting or mutating all of these domains is likely to interfere with overall folding of Staufen, thus confounding the interpretation of the results.
As an alternative approach to elucidating RNA-dependent vs RNA-independent roles of Staufen, we have now assessed the effect of the protein on in vitro motility of dynein-dynactin complexes formed in the presence of a constitutively active truncation of mammalian BicD2 (BicD2N). We find that Staufen partially inhibits motility of these 'DDB' complexes but not to the extent seen with the full length BicD in the presence of Egl and RNA (new Fig 2H, I and S3). As stated in the manuscript (lines 186-188) these data suggest that Staufen inhibits both the activation of dynein-dynactin motility by BicD proteins, as well as stimulation of this event by Egl and RNA. We believe these experiments provide significant new insight into Staufen's function. This finding is also incorporated into a new section of the Discussion dealing with potential roles of Staufen in addition to displacing Egl from RNPs.
A key question addressed is how does Staufen play a role in directing Oscar RNA localization to the posterior pole. The spatiotemporal control of Staufen at stage 9 seems to be a critical step. A number of experiments are performed to show that Staufen RNA enters the oocyte and accumulates to anterior pole through a process dependent on Egl (Fig. 7). -Definitive evidence is needed to show the role of 3'UTR of Stau and Egl binding. As it stands now, no evidence is presented to prove that delivery of staufen RNA via Egl, rather than dumping of Staufen protein into oocytes is the necessary trigger for the switch. It is well known that Staufen protein is also transported through ring canals to deliver Staufen into oocytes. There is no need to invoke an additional mechanism of Egl mediated staufen mRNA delivery. A key experiment is to perturb the Egl interaction with staufen 3'UTR and show this is a necessary component to impact oscar. Related to this comment, they should first perform biochemistry IP and PCR to demonstrate association of Egl with staufen RNA, and then somehow perturb this interaction to assess effects on oscar RNA localization. For example, is the 3'UTR of staufen RNA necessary for this mechanism? What if staufen RNA was ectopically localized in some inappropriate manner, for example localized to posterior pole? Would this prevent the switch of oscar RNA to move to posterior pole? The key question is: is it necessary that translation of Stau be coupled to Egl in order to drive the switch.
Mapping of the Egl-binding site in stau mRNA is a major undertaking requiring the production and evaluation of multiple new transgenic fly lines. We feel that this would constitute an entirely new study. Moreover, multiple lines of evidence already support a functional interaction between

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Egl and stau mRNA, notably the presence of Egl on stau RNPs (previously Fig. 7I, now Fig. 7F), the strongly impaired accumulation of stau mRNA in the oocyte of egl RNAi egg chambers, and the ability of Egl overexpression to reposition a subset of the stau mRNA population at the anterior cortex.
We have now performed new experiments and analyses to test the alternative hypothesis that Staufen protein is transported into the oocyte in the absence of stau mRNA transport. We find that disrupting Egl function with RNAi impairs localisation of both stau mRNA and protein in the proto-oocyte (new Figure 7A-D). As Egl has no known function in protein transport, these data argue against an RNA-independent mechanism for Staufen protein delivery. Moreover, we showed that both stau mRNA and Staufen are enriched in early oocytes lacking oskar mRNA, the main target of Staufen protein in the female germline. This result shows that Staufen protein is not appreciably transported from the nurse cells to the oocyte by hitchhiking on its RNA targets.
Whilst Mhlanga et al. 2009 did report transport of large GFP-Staufen particles through ring canals, the line used (matTub4>GFP-Staufen from the St Johnston lab, which was also used for our rescue experiments) is known to make protein aggregates which is not the case for the endogenous protein (Zimyanin et al., 2008 and our new Figures 7B and S7E-I) and are therefore likely to be artefactual. Neither we, nor previous studies (Little et al., 2015), detected endogenous Staufen protein in nurse cells.
Finally, the reviewer asks if coupling Staufen translation to Egl-mediated enrichment of stau mRNA in the oocyte is important: we showed in the original submission that strong overexpression of GFP-Staufen by Gal4 drivers leads to mislocalization of Staufen in the nurse cells of early egg-chambers, presumably due to saturation of the Egl-based transport machinery. In these egg-chambers, we observed defects in RNA enrichment in the primordial oocyte and defects in oogenesis, consistent with the need to exclude Staufen protein from the nurse cells.
These findings are now presented in new panels of the updated Figures 7 and S7, with the corresponding section of the manuscript revised accordingly (lines 410-420). We think that altogether these lines of evidence strongly support our model that Egl transports stau mRNA into the developing oocyte and that this process is pivotal for oskar RNA localization. **Minor comments** "Substantially more oskar mRNA was co-immunoprecipitated with Egl-GFP from extracts of egg-chambers expressing staufen RNAi compared to the control (Fig 3G). -This data is not shown in 3G, but rather only in Fig. S4H which needs quantitative analysis shown.

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This point stems from us calling out the wrong panel in the first submission; this has now been addressed, as described above. We apologize for the error.
"Addition of recombinant Staufen to the Egl, BicD, dynein and dynactin assembly mix significantly reduced the number of oskar mRNA transport events ( Fig. 2A and B)." -In Fig. 2A, the Y axis shows velocity not number of transport events  Staufen may also be required for the efficient release of the mRNA from the anterior cortex. This may reflect a role of Staufen in the coupling of the mRNA to the kinesin-dependent posterior transport pathway. This could be discussed as another aspect of the inhibition of dynein and handoff to kinesin. This is an interesting idea but it does not fit with our observation that Staufen depletion does not alter the association of oskar RNPs with kinesin-1 (originally Fig. 1C, now Fig. 1D). We do, however, now include in the Discussion a section on other ways, in addition to promoting Egl disassociation, that Staufen might orchestrate oskar mRNA transport. Here the authors demonstrate the competitive displacement of the RNA binding protein Egalitarian, which antagonizes dynein dependent localization at the anterior pole. This work done in this well characterized model of mRNA localization in Drosophila oocytes has broader implications for how the bidirectional transport of mRNAs is regulated in other polarized and highly differentiated cells, where very little is know about how mRNA transport direction might be regulated by opposing activities of kinesin and dynein motors. The strengths of this study are the integration of microscopy, biochemisty and genetic mutants to provide very nice experimental support for the two major aspects to the proposed model: 1) the competition between Staufen and Egl on oscar RNA which affects localization, 2) evidence for Egl mediated localization of staufen RNA into the oocyte as a key trigger for competitive displacement to bias localization of oscar RNA via kinesin. However, some additional experimental evidence is needed to solidify the conclusions and provide definitive support for this model, as discussed in other section. Thank you for submitting your revised manuscript entitled "An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport". The original reviewers have now assessed your revised manuscript and, as you can see, they are satisfied with the revisions. However, one reviewer did note that additional information in the legends would make the paper more streamlined and general copyediting would ensure the correct figures are referenced in the text. We would be happy to publish your paper in JCB pending final revisions to address this minor reviewer points. In your final revision, please ensure that you comply with our formatting guidelines (see details below).
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We apologize for these errors:  Figure 4F can be negative. Is it normalized to GFP-only control? Please include the related information in the legends. Another example is Figure 6A-C: how are BicD and p50 labeled? By UASp-BicD.eGFP and UAS-DCTN2-p50::GFP, respectively? If so, how are these transgenes driven? Why does osk mRNA have some mislocalization phenotype, especially in Figures 6A and 6B? All the information should be included in the Figure legends.
We have added the missing information to the figure legends as requested.
(4) Some figures have the error bars overlapping, making it really hard to distinguish, such as Figure 4E, Figure S5F, and Figure 6D-F. For Figure 4E, is there a statistically significant difference between control (blue) and BicC1/+ (purple)?
We agree with the reviewer that in some instances it was difficult to interpret the graphs due to overlapping error bars.
We have now introduced a slight offset in the x-axis between overlapping items in Figures 4-6 and S4 to address this issue.
In general, we only tested whether the mean of the distributions have a true difference to zero (alpha = 0.01, one sample ttest) but not the difference between conditions. In response to the reviewer's specific question, Egl association with oskar RNPs with ~4 copies of mRNA in control oocytes is not different from zero (indicated by triangles), whereas the same metric is significantly higher than zero in the BicD [1]/+ oocytes.
Some minor scientific questions: 1. Line 391-392, the authors claimed that "both Staufen and Staufen-GFP signal accumulated in the oocyte even in the absence of oskar mRNA (Fig S7A-C)". However, the endogenous Staufen level is much lower in the oskar mutant background ( Figure S7A') compared to the control ( Figure S7A). It is not consistent with the text. And I cannot find any quantification of this set of data.